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1.
Invest Ophthalmol Vis Sci ; 63(8): 5, 2022 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-35816045

RESUMO

Purpose: To spatially map aquaporin-5 (AQP5) expression in the bovine lens, molecularly characterize cytoplasmic AQP5-containing vesicles in the outer cortex, and elucidate AQP5 membrane trafficking mechanisms. Methods: Immunofluorescence was performed on bovine lens cryosections using AQP5, TOMM20, COX IV, calnexin, LC3B, Sec22ß, LIMP-2, and connexin 50 antibodies and the membrane dye CM-DiI. AQP5 plasma membrane insertion was defined via line expression profile analysis. Transmission electron microscopy (TEM) was performed on bovine lens sections to examine cytoplasmic organelle morphology and subcellular localization in cortical fiber cells. Bovine lenses were treated with 10-nM bafilomycin A1 or 0.1% dimethyl sulfoxide vehicle control for 24 hours in ex vivo culture to determine changes in AQP5 plasma membrane expression. Results: Immunofluorescence analysis revealed cytoplasmic AQP5 expression in lens epithelial cells and differentiating fiber cells. In the lens cortex, complete AQP5 plasma membrane insertion occurs at r/a = 0.951 ± 0.005. AQP5-containing cytoplasmic vesicles are spheroidal in morphology with linear extensions, express TOMM20, and contain LC3B and LIMP-2, but not Sec22ß, as fiber cells mature. TEM analysis revealed complex vesicular assemblies with congruent subcellular localization to AQP5-containing cytoplasmic vesicles. AQP5-containing cytoplasmic vesicles appear to dock with the plasma membrane. Bafilomycin A1 treatment reduced AQP5 plasma membrane expression by 27%. Conclusions: AQP5 localizes to spheroidal, linear cytoplasmic vesicles in the differentiating bovine lens fiber cells. During fiber cell differentiation, these vesicles incorporate LC3B and presumably fuse with LIMP-2-positive lysosomes. Our data suggest that AQP5 to the plasma membrane through lysosome-associated unconventional protein secretion, a novel mechanism of AQP5 trafficking.


Assuntos
Aquaporina 5 , Cristalino , Animais , Aquaporina 5/metabolismo , Bovinos , Membrana Celular/metabolismo , Córtex do Cristalino/metabolismo , Cristalino/metabolismo , Transporte Proteico
2.
Ophthalmic Genet ; 42(6): 744-746, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34282983

RESUMO

INTRODUCTION: Cataract is a major condition characterized by ocular lens opacification, resulting from alteration in the lens architecture, lens proteins or both. It is responsible for about one-third of infants' blindness worldwide. Variants in the FYCO1 gene have been associated with autosomal recessive infantile cataract. MATERIAL AND METHODS: We conducted whole exome sequencing (WES) in a nine months old male patient who was referred for genetic investigation because of infantile cataract. WES analysis revealed the presence of a homozygous pathogenic variant (c.2365C>T) in exon 8 of the FYCO1 gene. RESULTS AND DISCUSSION: This is the first report on a Lebanese infant with infantile cataract and cortical atrophy which was not previously reported, resulting from a novel homozygous FYCO1 variant; thus expanding the clinical phenotypic spectrum of FYCO1 involvement.


Assuntos
Catarata/genética , Códon sem Sentido/genética , Córtex do Cristalino/patologia , Proteínas Associadas aos Microtúbulos/genética , Mutação , Atrofia , Catarata/congênito , Catarata/diagnóstico , Consanguinidade , Éxons/genética , Genes Recessivos , Homozigoto , Humanos , Lactente , Masculino , Linhagem , Reação em Cadeia da Polimerase
3.
Ophthalmic Genet ; 42(6): 773-779, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34310258

RESUMO

BACKGROUND: Whereas crystals deposit in the retina, the cornea and limbus in Bietty corneo-retinal dystrophy (BCD) is now well established and documented, only two published cases report their findings in the lens and no cases deep in the lens cortex. MATERIAL AND METHODS: Four consecutive adult patients from three different unrelated families presenting lens crystals associated with advanced genetically confirmed BCD were enrolled with advanced disease and long follow up (>12 years). Demographics, visual acuity, slit lamp biomicroscopy, lens and posterior pole photography, optical coherence tomography (OCT), autofluorescence, and screening for CYP4V2 type of mutation were performed. The setting was Jules Gonin Eye Hospital, Switzerland, between 1.1 2013 and 1.11. 2019. RESULTS: All patients were European women. The ages ranged from 40 to 81 years. Best Snellen visual acuity ranged from light perception to 1.0. All patients presented with limbus and retinal crystals deposit that disappeared over time and the development of severe chorioretinal atrophy. With long-term follow up, multiple crystal-like deposits appeared in the anterior, posterior lens capsule and cortex. All patients, but one, had homozygous or compound heterozygous mutations in CYP4V2 gene. CONCLUSIONS: To the best of our knowledge, there are no published cases of crystal deposits in the cortex of the lens of patients diagnosed with BCD associated with CYP4V2 gene mutation. This could be a feature of advanced BCD, and their presence in the lens cortex questions the hypothesis of floating deposits from posterior pole although their exact etiology remains to be determined.


Assuntos
Distrofias Hereditárias da Córnea/diagnóstico , Distrofias Hereditárias da Córnea/genética , Família 4 do Citocromo P450/genética , Córtex do Cristalino/patologia , Mutação , Doenças Retinianas/diagnóstico , Doenças Retinianas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Distrofias Hereditárias da Córnea/fisiopatologia , Cristalização , Feminino , Humanos , Pessoa de Meia-Idade , Imagem Óptica , Doenças Retinianas/fisiopatologia , Microscopia com Lâmpada de Fenda , Tomografia de Coerência Óptica , Acuidade Visual/fisiologia
4.
Rev. cuba. oftalmol ; 34(2): e1018, 2021. tab, graf
Artigo em Espanhol | LILACS, CUMED | ID: biblio-1341461

RESUMO

La catarata comprende la opacidad del cristalino, la cual puede afectar la corteza y el núcleo subcapsular anterior y posterior de manera progresiva, secundario a la acumulación de proteínas dañadas a este nivel, con pérdida del equilibrio entre la producción y la eliminación de las especies reactivas libres de oxígeno. La importancia de retrasar o identificar marcadores específicos, además de promover un nuevo blanco terapéutico, también es motivo de análisis y de estudio en diferentes líneas de investigación. Se realizó una revisión de la literatura del 01 de enero al 20 de julio del año 2020. Se utilizaron metabuscadores en inglés y español de PUBMED, INFOMED, CLINICALKEY, LILACS, EBSCO, SCIELO, PRISMA y UPTODATE, con el objetivo de identificar la nueva evidencia científica relacionada con el estrés oxidativo y su participación en la formación de la catarata. La barrera del cristalino funciona como un medio de intercambio entre diferentes moléculas, lo que impide el paso de antioxidantes al núcleo y provoca su opacificación. Las mitocondrias a nivel de la corteza del cristalino permiten la remoción de oxígeno. Posteriormente la fosforilación oxidativa forma radicales libres de superóxido que, de manera natural, con el paso del tiempo se acumulan a este nivel. Con la edad, la homeostasis adaptativa pierde la capacidad de responder ante los cambios de estrés oxidativo, por lo que el uso de antioxidantes -de manera profiláctica e intencionada- puede cambiar el destino último para esta patología. La falta de equilibrio en los procesos de óxido-reducción es responsable de la formación de la catarata(AU)


Cataract comprises opacification of the crystalline lens, which may progressively affect the cortex and the anterior subcapsular nucleus, secondary to accumulation of damaged proteins on this level, with loss of balance between production and elimination of free reactive oxygen species. The importance of delaying or identifying specific markers, as well as promoting a new therapeutic target, is the object of study and analysis of a variety of research lines. A review was conducted of the literature published from 1 January to 20 July 2020. Use was made of PubMed, Infomed, Clinical Key, Lilacs, EBSCO, SciELO, Prisma and UpToDate metasearch engines in English and Spanish to identify new scientific evidence about oxidative stress and its involvement in cataract formation. The crystalline lens barrier serves as a medium for exchange between various molecules, preventing entrance of antioxidants into the nucleus, which results in opacification. Mitochondria on the crystalline lens cortex allow oxygen removal. Oxidative phosphorylation then forms free superoxide radicals which naturally accumulate on this level with the passing of time. With aging, adaptive homeostasis loses its ability to respond to oxidative stress changes, but the prophylactic, targeted use of antioxidants may change the ultimate fate of this condition. Lack of balance in oxidation-reduction processes is the cause of cataract formation(AU)


Assuntos
Humanos , Oxirredução , Catarata/etiologia , Espécies Reativas de Oxigênio , Homeostase , Córtex do Cristalino , Literatura de Revisão como Assunto
5.
Exp Eye Res ; 206: 108536, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33716012

RESUMO

Eye lens membranes are complex biological samples. They consist of a variety of lipids that form the lipid bilayer matrix, integral proteins embedded into the lipid bilayer, and peripheral proteins. This molecular diversity in membrane composition induces formation of lipid domains with particular physical properties that are responsible for the maintenance of proper membrane functions. These domains can be, and have been, effectively described in terms of the rotational diffusion of lipid spin labels and oxygen collision with spin labels using the saturation recovery (SR) electron paramagnetic resonance method and, now, using stretched exponential function for the analysis of SR signals. Here, we report the application of the stretched exponential function analysis of SR electron paramagnetic resonance signals coming from cholesterol analog, androstane spin label (ASL) in the lipid bilayer portion of intact fiber cell plasma membranes (IMs) isolated from the cortex and nucleus of porcine eye lenses. Further, we compare the properties of these IMs with model lens lipid membranes (LLMs) derived from the total lipids extracted from cortical and nuclear IMs. With this approach, the IM can be characterized by the continuous probability density distribution of the spin-lattice relaxation rates associated with the rotational diffusion of a spin label, and by the distribution of the oxygen transport parameter within the IM (i.e., the collision rate of molecular oxygen with the spin label). We found that the cortical and nuclear LLMs possess very different, albeit homogenous, spin lattice relaxation rates due to the rotational diffusion of ASL, indicating that the local rigidity around the spin label in nuclear LLMs is considerably greater than that in cortical LLMs. However, the oxygen transport parameter around the spin label is very similar and slightly heterogenous for LLMs from both sources. This heterogeneity was previously missed when distinct exponential analysis was used. The spin lattice relaxation rates due to either the rotational diffusion of ASL or the oxygen collision with the spin label in nuclear IMs have slower values and wider distributions compared with those of cortical IMs. From this evidence, we conclude that lipids in nuclear IMs are less fluid and more heterogeneous than those in cortical membranes. Additionally, a comparison of properties of IMs with corresponding LLMs, and lipid and protein composition analysis, allow us to conclude that the decreased lipid-to-protein ratio not only induces greater rigidity of nuclear IMs, but also creates domains with the considerably decreased and variable oxygen accessibility. The advantages and disadvantages of this method, as well as its use for the cluster analysis, are discussed.


Assuntos
Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Córtex do Cristalino/metabolismo , Núcleo do Cristalino/metabolismo , Lipídeos de Membrana/metabolismo , Animais , Colesterol/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Córtex do Cristalino/citologia , Núcleo do Cristalino/citologia , Bicamadas Lipídicas/metabolismo , Fluidez de Membrana , Modelos Animais , Marcadores de Spin , Suínos
6.
Invest Ophthalmol Vis Sci ; 62(1): 12, 2021 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-33427852

RESUMO

Purpose: Human cortical opacities are most commonly accompanied by changes in lens fiber structure in the equatorial region at the lens nucleus-cortex interface. Cortex and nucleus have different elastic properties, which change with age. We therefore subjected ex vivo lenses to simulated accommodation and studied the internal deformations to better understand the mechanism of cortical cataract formation. Methods: Nine human donor lenses (33-88 years old) were tested using a bespoke radial stretching device for anterior eye segments. Seven of the lenses exhibited cortical cataracts. The other two lenses, without cataract, were used as controls. Frontal and cross-sectional images of the lens obtained during stretching facilitated measurements on equatorial lens diameter and central lens thickness in the stretched and unstretched states. Results: Stretching caused the lens equatorial diameter to increase in all cases. Conversely, the lens central thickness showed no systematic variation during stretching. For four of the lenses with cortical cataract, ruptures were observed during stretching at the nucleus-cortex boundary adjacent to the cortical cataracts. Ruptures were not observed in the control lenses or in the three other lenses with cortical cataract. Conclusions: Internal ruptures can occur in aged ex vivo lenses subjected to simulated disaccommodation. These ruptures occur at the nucleus-cortex interface; at this location, a significant stiffness discontinuity is expected to develop with age. It is hypothesized that ruptures occur in in vivo lenses during accommodation-or attempted accommodation.


Assuntos
Acomodação Ocular/fisiologia , Catarata/fisiopatologia , Córtex do Cristalino/fisiopatologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Fenômenos Biomecânicos , Catarata/diagnóstico por imagem , Estudos Transversais , Humanos , Córtex do Cristalino/diagnóstico por imagem , Pessoa de Meia-Idade , Fotografação , Ruptura Espontânea , Estresse Fisiológico , Doadores de Tecidos
7.
J Toxicol Sci ; 45(4): 201-218, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32238695

RESUMO

TP0446131, developed as an antidepressant agent, was found to cause lenticular opacity in a 13-week repeated-dose study in dogs. Histopathologically, the lenticular opacity was observed as a degeneration of the lens fibers, characterized by irregularity in the ordered arrangement of the fibers which is necessary to maintain the transparency of the lens, and was considered to manifest clinically as cataract. To evaluate the development mechanism of the lenticular opacity, the chemical constituents of the lens, which is known to be associated with the development of cataract, were examined. The results of liquid chromatography-tandem mass spectrometry analysis revealed an increase in the amplitudes of 3 unknown peaks in a dose- and time-dependent manner in the lens, with no remarkable changes in the other chemical components tested. In addition, the content of cholesterol, alterations of which have been reported to be associated with cataract, remained unchanged. The mass spectral data and chromatographic behavior of the 3 peaks indicated that these peaks corresponded to sterol-related substances, and that one of them was 7-dehydrocholesterol, a precursor of cholesterol biosynthesis. This finding suggested that TP0446131 exerts some effects on the cholesterol biosynthesis pathway, which could be involved in the development of the cataracts. Furthermore, increases in the levels of these sterol-related substances were also detected in the serum, and were, in fact, noted prior to the onset of the cataract, suggesting the possibility that these substances in the serum could be used as potential safety biomarkers for predicting the onset of cataract induced by TP0446131.


Assuntos
Antidepressivos/efeitos adversos , Catarata/induzido quimicamente , Desidrocolesteróis/metabolismo , Córtex do Cristalino/metabolismo , Córtex do Cristalino/patologia , Biomarcadores/sangue , Catarata/diagnóstico , Catarata/metabolismo , Cromatografia Líquida , Desidrocolesteróis/sangue , Relação Dose-Resposta a Droga , Humanos , Masculino , Espectrometria de Massas em Tandem
8.
Medicine (Baltimore) ; 98(39): e17054, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31574802

RESUMO

RATIONALE: To report the visual status and results of phacoemulsification cataract surgery in a young patient with Alport syndrome associated with bilateral anterior lenticonus. The milestone of this report is the use of anterior segment optical coherence tomography (AS-OCT) to confirm the central protrusion of the anterior surface of the crystalline lens. PATIENT CONCERNS: A 23-year-old young woman presented with severe progressive visual loss in both eyes, which started several years ago. DIAGNOSES: Refractive status was indicative of high myopia with astigmatism and vision was not improved with optimal correction to better than 0.1 in the right eye and 0.2 in the left eye (visual acuities given in decimal notation). Slit-lamp examination showed transparent cornea, anterior lenticonus and posterior sub-capsular cataract in both eyes. The classical appearance of oil droplet was evident using retro-illumination on the slit lamp. INTERVENTIONS: The natural lenses were replaced with intraocular lens (IOL). OUTCOMES: An excellent refractive status achieved associated with an uncorrected distance visual acuity 0.9 and 0.8 in the right and left eye, respectively. LESSONS: AS-OCT is a valuable device for confirming the budging of the anterior crystalline lens surface.


Assuntos
Cápsula do Cristalino/patologia , Cápsula do Cristalino/cirurgia , Córtex do Cristalino/patologia , Córtex do Cristalino/cirurgia , Nefrite Hereditária/patologia , Nefrite Hereditária/cirurgia , Facoemulsificação , Feminino , Humanos , Cápsula do Cristalino/diagnóstico por imagem , Córtex do Cristalino/diagnóstico por imagem , Nefrite Hereditária/diagnóstico por imagem , Tomografia de Coerência Óptica , Baixa Visão/diagnóstico por imagem , Baixa Visão/etiologia , Baixa Visão/cirurgia , Adulto Jovem
9.
Exp Eye Res ; 178: 72-81, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30278157

RESUMO

Four purported lipid domains are expected in plasma membranes of the eye lens fiber cells. Three of these domains, namely, bulk, boundary, and trapped lipids, have been detected. The cholesterol bilayer domain (CBD), which has been detected in lens lipid membranes prepared from the total lipids extracted from fiber cell plasma membranes, has not yet been detected in intact fiber cell plasma membranes. Here, a saturation-recovery electron paramagnetic resonance spin-labeling method has been developed that allows identification of CBDs in intact fiber cell plasma membranes of eye lenses. This method is based on saturation-recovery signal measurements of the cholesterol-analog spin label located in the lipid bilayer portion of intact fiber cell membranes as a function of the partial pressure of molecular oxygen with which the samples are equilibrated. The capabilities and limitations of this method are illustrated for intact cortical and nuclear fiber cell plasma membranes from porcine eye lenses where CBDs were detected in porcine nuclear intact membranes for which CBDs were also detected in lens lipid membranes. CBDs were not detected in porcine cortical intact and lens lipid membranes. CBDs were detected in intact membranes isolated from both cortical and nuclear fiber cells of lenses obtained from human donors. The cholesterol content in fiber cell membranes of these donors was always high enough to induce the formation of CBDs in cortical as well as nuclear lens lipid membranes. The results obtained for intact membranes, when combined with those obtained for lens lipid membranes, advance our understanding of the role of high cholesterol content and CBDs in lens biology, aging, and/or cataract formation.


Assuntos
Membrana Celular/química , Colesterol/química , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Córtex do Cristalino/química , Núcleo do Cristalino/química , Bicamadas Lipídicas/química , Lipídeos de Membrana/química , Animais , Interações Hidrofóbicas e Hidrofílicas , Fluidez de Membrana , Marcadores de Spin , Suínos
11.
J Cataract Refract Surg ; 44(6): 677-679, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29909959

RESUMO

We describe the technique of second-wave hydrodissection (the first wave being the initial cortical cleaving hydrodissection) performed after the removal of the cataract nucleus in femtosecond laser-assisted cataract surgery. After femtosecond laser application, the cortex is typically found adhered to the anterior capsule. Under high magnification, a steady stream of a balanced salt solution is directed toward the anterior capsule using a hydrodissection cannula. Full cleavage of the remaining cortex is observed by noting the appearance of a dark inner circle by the capsulotomy edge once the balanced salt solution wave has separated the cortex from the capsule. Irrigation/aspiration (I/A) of the cortical remains after the second wave is faster than I/A without this step in femtosecond laser-assisted cataract surgery.


Assuntos
Extração de Catarata/métodos , Terapia a Laser/métodos , Córtex do Cristalino/cirurgia , Capsulorrexe/métodos , Humanos , Período Pós-Operatório
12.
Exp Eye Res ; 171: 131-141, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29571628

RESUMO

Although it is well-known that protein turnover essentially stops in mature lens fiber cells, mapping out the ensuing protein degradation and its effects on lens function over time remains challenging. In particular, isomerization is a common, spontaneous post-translational modification that occurs over long timescales and generates products invisible to most analytical methods. Nevertheless, isomerization can significantly impact protein structure, function, and solubility, which are all necessary to maintain clarity and proper refractive index within the lens. Herein, we examine the degree of isomerization occurring in crystallin proteins in the human eye lens as a function of both age and location within the lens. A novel mass spectrometric technique leveraging radical chemistry enables detailed characterization of proteins extracted from the cortex and nucleus of the lens. It is observed that the degree of isomerization increases significantly between the cortex and nucleus and between water-soluble and water-insoluble fractions. Interestingly, the abundance of L-isoAsp is low in the water-soluble cortex despite being the dominant product generated by isomerization of Asp in vitro, suggesting that Protein L-isoaspartyl methyltransferase (PIMT) is active in the cortex and suppresses the accumulation of L-isoAsp. The abundance of L-isoAsp increases dramatically in the nucleus, revealing that PIMT activity decreases over time in the center of the lens. In addition, the growth of L-isoAsp in the nuclear fraction suggests protein isomerization continues within the nucleus, despite the fact that most of the protein within the nucleus has become insoluble. Additionally, it is demonstrated that sequential Asp residues lead to isomerization hotspots in human crystallin proteins and that the isomerization profiles for αA and αB crystallin are notably different. Although αA is more prone to isomerization, αB loses solubility more rapidly upon modification. These differences are likely related to the distribution of Asp residues within αA and αB, which are in turn connected to refractive index. The high Asp content of αA is a hazard in terms of isomerization and aging, but it serves to enhance the refractive index of αA relative to αB, and may explain why αA is only found in the eye.


Assuntos
Córtex do Cristalino/enzimologia , Núcleo do Cristalino/enzimologia , Proteína D-Aspartato-L-Isoaspartato Metiltransferase/metabolismo , alfa-Cristalinas/química , Adulto , Idoso , Envelhecimento/fisiologia , Cromatografia Líquida de Alta Pressão , Humanos , Isomerismo , Pessoa de Meia-Idade , Oligopeptídeos/química , Processamento de Proteína Pós-Traducional , Espectrometria de Massas em Tandem
13.
Clin Exp Optom ; 101(1): 64-68, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-28370270

RESUMO

BACKGROUND: The shape of the human lens changes from almost spherical at birth to ellipsoid due to a decrease in sagittal thickness and an increase in equatorial diameter during the first two decades of life. Both dimensions increase thereafter. This study was undertaken to determine the reason for the change. METHODS: Published refractive index gradients, from 20 lenses aged from seven to 82 years, were used to calculate the protein contents of concentric shells of fibre cells in human lenses. The boundaries of nuclear cores containing from 2.5 to 45 mg, in 2.5 mg increments, were determined from the isoindicial shells. Cortex thickness was determined from the distance between the 30 mg nuclear boundary and the capsule. RESULTS: The sagittal thickness of every nuclear core decreased until age 40 years and remained constant thereafter. Over the same time frame, the equatorial diameter of the cores containing up to 30 mg of protein increased, while those of cores larger than 30 mg decreased. The volumes of the cores decreased and their shapes changed from near spherical to spheroidal. Equatorial and sagittal cortex thickness increased linearly with age at 0.0082 mm per year. The anterior sagittal cortex was 0.23 mm larger than the posterior and the equatorial cortex was 0.62 mm greater. CONCLUSIONS: Changes in lens shape observed during the first two decades of life are due to remodelling and compaction of the 30 mg nuclear core. Cortex growth is linear throughout life.


Assuntos
Envelhecimento , Córtex do Cristalino/anatomia & histologia , Núcleo do Cristalino/anatomia & histologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Humanos , Córtex do Cristalino/crescimento & desenvolvimento , Núcleo do Cristalino/crescimento & desenvolvimento , Pessoa de Meia-Idade , Tamanho do Órgão , Adulto Jovem
14.
Indian J Ophthalmol ; 65(1): 59-61, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-28300744

RESUMO

We describe here a surgical technique of removing the remaining cortex after phacoemulsification without performing the conventional irrigation/aspiration (I/A) procedure. In this technique, the remaining cortex attached to the posterior capsule was separated and dissected into several pieces by continuous irrigation with balanced salt solution, which was supplied through a syringe attached to a bent, blunt-tip needle. Approximately, 10 s of manual irrigation separated most of the remaining cortex from the posterior capsule. Then, the capsular bag was inflated with an ophthalmic viscoelastic device (OVD), and this pushed the separated cortex toward the capsular fornix mechanically. An intraocular lens was inserted into the capsular bag, following which the remaining cortex and OVD were removed concomitantly using an automated I/A handpiece. This technique is a simple and easy maneuver to remove the cortex from all areas, including the subincisional area, and reduce the possibility of a posterior capsule tear.


Assuntos
Extração de Catarata/métodos , Córtex do Cristalino/cirurgia , Humanos , Sucção/métodos , Irrigação Terapêutica/métodos
15.
Acta Ophthalmol ; 95(8): 834-838, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28083904

RESUMO

PURPOSE: The aim of this study was to investigate in which part of the lens in vivo exposure to subthreshold dose of UVR-B radiation induces apoptosis. METHODS: Twenty 6-week-old female albino Sprague-Dawley rats were exposed to subthreshold dose (1 kJ/m2 ) of UVR-B unilaterally and killed at 120 hr after exposure. Lenses were enucleated and dissected on three regions: the lens epithelium, the cortex and the nucleus. The lens nucleus then was removed. Apoptosis markers p53 and caspase 3 were used to study apoptosis in the lens regions. qRT-PCR and Western blot were utilized to analyse the lenses. RESULTS: TP53 and CASP3 mRNA expressions are increased in exposed lenses, both in the lens epithelium and in the cortex regions, in relation to non-exposed lenses. Expression of p53 protein is increased in exposed lens epithelium in relation to non-exposed lens epithelium. Caspase 3 protein is expressed in exposed lens epithelial cells, while it is not expressed in non-exposed lens epithelial cells. p53 and caspase 3 proteins are not expressed in either exposed nor non-exposed lens fibre cells. CONCLUSION: Exposure to UVR-B increases mRNA transcription of apoptosis marker p53 in vivo in both regions of the lens and of apoptosis marker caspase 3 in the lens cortex. Exposure to UVR-B increases p53 and caspase 3 proteins expression just in the lens epithelium. In vivo exposure to subthreshold dose of UVR-B induces apoptosis in the lens epithelial cells and does not in the lens fibre cells.


Assuntos
Apoptose/efeitos da radiação , Catarata/diagnóstico , Células Epiteliais/patologia , Córtex do Cristalino/patologia , Lesões Experimentais por Radiação/patologia , Raios Ultravioleta/efeitos adversos , Animais , Catarata/etiologia , Relação Dose-Resposta à Radiação , Células Epiteliais/efeitos da radiação , Feminino , Córtex do Cristalino/efeitos da radiação , Lesões Experimentais por Radiação/complicações , Ratos , Ratos Sprague-Dawley , Espalhamento de Radiação
17.
Invest Ophthalmol Vis Sci ; 57(11): 4721-32, 2016 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-27607418

RESUMO

PURPOSE: Glutathione S-Transferase Mu 3 (GSTM3) protects the lens from oxidative stress that contributes to age-related cataract (ARC) formation. We examined the expression and epigenetics of GSTM3 in lens epithelial cells (LECs) and lens cortex of ARC, and investigated the potential role of molecular changes in ARC pathogenesis. METHODS: This study included 120 ARCs and 40 controls. Expression of GSTM3, DNA methylation, and histone modification were assessed by quantificational real-time PCR, Western blot, bisulfite-sequencing PCR, pyrosequencing, and chromatin immunoprecipitation assay. Human lens epithelial (HLE) cell lines, SRA01/04 and HLEB3, were served as an in vitro model to observe the relationship between epigenetic status and GSTM3 expression. Potential transcription factors binding to GSTM3 promoter were detected by electrophoretic mobility shift assay. RESULTS: Expression of GSTM3 decreased in ARC lens tissues compared to that in the controls, which correlated with the hypermethylation of GSTM3 promoter. Lower level of GSTM3 was detected in HLEB3 than in SRA01/04, while HLEB3 displayed hypermethylation of GSTM3 and SRA01/04 did not. Compared to SRA01/04, HLEB3 displayed lower acetylated H3 and higher trimethylated H3K9 levels. After treatment with DNA methyltransferase inhibitor or histone deacetylase inhibitor, HLEB3 had an increased GSTM3 expression. Methylation of GSTM3 promoter abrogated the potential transcription factor binding. The GSTM3 expression declined in hydrogen peroxide-treated HLE cell lines. CONCLUSIONS: Expression of GSTM3 might be regulated by epigenetic changes in lens tissue. Hypermethylation in GSTM3 promoter and altered histone modification might have a role in the ARC formation. The results provided a potential strategy of ARC management by manipulating epigenetic changes.


Assuntos
Envelhecimento/genética , Catarata/genética , DNA/genética , Epigenômica/métodos , Regulação da Expressão Gênica , Glutationa Transferase/genética , Córtex do Cristalino/metabolismo , Catarata/metabolismo , Catarata/patologia , Células Cultivadas , Metilação de DNA , Feminino , Glutationa Transferase/biossíntese , Humanos , Córtex do Cristalino/patologia , Masculino , Pessoa de Meia-Idade , Estresse Oxidativo , Regiões Promotoras Genéticas , Reação em Cadeia da Polimerase em Tempo Real
18.
Invest Ophthalmol Vis Sci ; 57(10): 4108-14, 2016 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-27537260

RESUMO

PURPOSE: To quantify protein changes in the morphologically distinct remodeling zone (RZ) and adjacent regions of the human lens outer cortex using spatially directed quantitative proteomics. METHODS: Lightly fixed human lens sections were deparaffinized and membranes labeled with fluorescent wheat germ agglutinin (WGA-TRITC). Morphology directed laser capture microdissection (LCM) was used to isolate tissue from four distinct regions of human lens outer cortex: differentiating zone (DF), RZ, transition zone (TZ), and inner cortex (IC). Liquid chromatography-tandem mass spectrometry (LC-MS/MS) of the plasma membrane fraction from three lenses (21-, 22-, and 27-year) revealed changes in major cytoskeletal proteins including vimentin, filensin, and phakinin. Peptides from proteins of interest were quantified using multiple reaction monitoring (MRM) mass spectrometry and isotopically-labeled internal peptide standards. RESULTS: Results revealed an intermediate filament switch from vimentin to beaded filament proteins filensin and phakinin that occurred at the RZ. Several other cytoskeletal proteins showed significant changes between regions, while most crystallins remained unchanged. Targeted proteomics provided accurate, absolute quantification of these proteins and confirmed vimentin, periplakin, and periaxin decrease from the DF to the IC, while filensin, phakinin, and brain acid soluble protein 1 (BASP1) increase significantly at the RZ. CONCLUSIONS: Mass spectrometry-compatible fixation and morphology directed laser capture enabled proteomic analysis of narrow regions in the human lens outer cortex. Results reveal dramatic cytoskeletal protein changes associated with the RZ, suggesting that one role of these proteins is in membrane deformation and/or the establishment of ball and socket joints in the human RZ.


Assuntos
Cristalinas/metabolismo , Filamentos Intermediários/metabolismo , Córtex do Cristalino/metabolismo , Proteômica/métodos , Adulto , Animais , Citoesqueleto/metabolismo , Feminino , Humanos , Córtex do Cristalino/citologia , Masculino , Suínos , Espectrometria de Massas em Tandem , Adulto Jovem
19.
Genet Mol Res ; 15(2)2016 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-27323124

RESUMO

The aim of this study was to observe the proliferation of, and cell-cycle changes in, the human lens epithelial cell line HLEC after Toll-like receptor 4 (TLR4) gene silencing. HLEC cells were transfected with four TLR4-short hairpin RNA (shRNA) lentiviral vectors or the control lentivirus (pGCL-GFP-shRP-1, -2, -3, -4, NC). TLR4 silencing was verified in these cells 96 h post-transfection using real-time polymerase chain reaction and western blot. We also observed the change in number of pGCL-GFP-shRP-4-transfected HLEC cells with silenced TLR4 (multiplicity of infection = 10). Cell proliferation was analyzed 48 h after transfection by a standard Cell Counting Kit-8 (CCK-8) assay, and the cell cycle changes were detected by flow cytometry. The number of cells with silenced TLR4 decreased with time. The decrease in TLR4 expression led to decelerated cell proliferation. Cells with silenced TLR4 (for 48 h) were arrested in the G1 phase; that is, the cell cycle was prolonged and cell division was decelerated. Lentivirus-mediated RNA interference effectively silenced TLR4 expression in HLEC cells, which decelerated their proliferation rate and extended the cell cycle.


Assuntos
Proliferação de Células/genética , RNA Interferente Pequeno/genética , Receptor 4 Toll-Like/genética , Transfecção/métodos , Apoptose/genética , Ciclo Celular/genética , Linhagem Celular , Células Epiteliais/patologia , Inativação Gênica , Humanos , Córtex do Cristalino/metabolismo , Córtex do Cristalino/patologia , Lentivirus/genética , Receptor 4 Toll-Like/antagonistas & inibidores
20.
PLoS One ; 11(2): e0149249, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26890694

RESUMO

PURPOSE: To assess nuclear and cortical opacities through the objective analysis of Scheimpflug images, and to check the correlation with the Lens Opacity Classification System III (LOCS III). METHODS: Nuclear and cortical opacities were graded according to the LOCS III rules after pupil dilation. The maximum and average pixel intensity values along an elliptical mask within the lens nucleus were taken to analyse nuclear cataracts. A new metric based on the percentage of opaque pixels within a region of interest was used to analyse cortical cataracts. The percentage of opaque pixels was also calculated for half, third and quarter areas from the region of interest's periphery. RESULTS: The maximum and average intensity values along the nucleus were directly proportional to the LOCS III grade: The larger the LOCS III value, the larger maximum and average intensity ones. These metrics showed a positive and significant correlation with the LOCS grade: The larger the LOCS grade, the higher was percentage of opaque pixels along the cortex within the same mask's size. This metric showed a significant correlation to the LOCS grade. CONCLUSION: The metrics used to assess nuclear opacities showed good correlation with the LOCS III. The percentage of opaque pixels showed to be a useful metric to measure objectively the severity of the cortical opacity. These metrics could be implemented in an algorithm to detect and grade lens opacities automatically and objectively.


Assuntos
Catarata/diagnóstico , Diagnóstico por Imagem/métodos , Técnicas de Diagnóstico Oftalmológico , Córtex do Cristalino/patologia , Núcleo do Cristalino/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Catarata/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Índice de Gravidade de Doença , Adulto Jovem
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